Fatigue after traumatic brain injury : exploring novel methods for diagnosis and treatment

(1)

Fatigue after

Traumatic Brain Injury

Exploring Novel Methods for

Diagnosis and Treatment

(2)

Responsible publisher under Swedish law: the Dean of the Medical Faculty This work is protected by the Swedish Copyright Legislation (Act 1960:729) Dissertation for PhD

ISBN: 978-91-7601-979-5 ISSN: 0346-6612

New Series NO: 2000

Cover photo: Arnica Desiatnik Bäckström

Electronic version available at: http://umu.diva-portal.org/ Printed by: UmU Print Service, Umeå University

(3)

(4)

(5)

(6)

Abstract

Background

Traumatic brain injury (TBI) is one of the most common causes of disability and mortality. While some patients recover quickly, especially at the mild side of the injury severity continuum, many will experience symptoms for years to come. In this chronic phase, patients report a wide array of symptoms, where fatigue is one the most common. This fatigue makes huge impact in several areas of these patients’ lives. Despite the prevalence of fatigue after TBI, the underlying mechanisms are unclear. Further, there are no standardized way for assessment and diagnosis, and there are no treatments with satisfying empirical support. The aim of this thesis was to examine the effects of the novel compound OSU6162 on fatigue in patients with TBI, and to explore functional and structural brain imaging correlates of fatigue after TBI.

Methods

Studies I and III were based on a placebo-controlled, double-blinded clinical trial examining the effects of the monoaminergic stabilizer OSU6162 on fatigue in patients in the chronic phase of traumatic brain injury. In study I, self-assessment scales of fatigue and neuropsychological tests were used as outcomes, while functional magnetic resonance imaging (fMRI) blood-oxygen-level dependent (BOLD) signal was the primary outcome in study III. Studies II and IV used cross-sectional designs, comparing patients with TBI with age- and gender matched healthy controls. Study II examined whether fMRI BOLD signal could be used to detect and diagnose fatigue in patients with TBI, and study IV whether white matter hyperintensities (WMH) contribute to lower cognitive functioning and presence of fatigue after TBI.

Results

Study I revealed no effects of OSU6162 during 28 days of treatment at maximum doses of 15 mg twice daily on measures of fatigue or any other outcome. The results from study II indicated that fatigue after TBI is linked to alterations in striato-thalamic-cortical loops, and suggested that fMRI could be a promising technique to use in the diagnosis of fatigue after TBI. In study III the results revealed effects of treatment in the right occipitotemporal and orbitofrontal cortex. In these areas, the BOLD response was normalized in the OSU6162 group as compared to healthy controls, while the placebo group showed a steady low activity in these areas. The regional effects were located outside the network shown to be linked to fatigue in study II, which might explain why there were no

(9)

effects on fatigue after treatment with OSU6162 in study I. Study IV showed that WMH lesions increased with increased TBI severity, but the presence and extent of lesions did not explain lower neuropsychological functioning or fatigue in subjects with previous TBI.

Conclusions

In summary, although no effects on fatigue after treatment with OSU6162 were seen, the results provide support to the theory that fatigue after TBI is linked to alterations in striato-thalamic-cortical loops, and on how fatigue after TBI could be assessed or diagnosed using fMRI. Structural damage within white matter was however not related to fatigue.

(10)

Abbreviations

BOLD; Blood-oxygen-level dependent CBT; Cognitive Behavioral Therapy CT; Computed Tomography

D-KEFS; Delis-Kaplan Executive Functions System DAI; Diffuse Axonal Injury

ECG; Electrocardiography

fMRI; Functional Magnetic Resonance Imaging FSS; Fatigue Severity Scale

GCS; Glasgow Coma Scale

HAD; Hospital Anxiety and Depression Scale

IVA; Integrated Visual and Auditory Continuous Performance Test MFS; Mental Fatigue Scale

MNI; Montreal Neurological Institute MRI; Magnetic Resonance Imaging

mSDMT; modified Symbol Digit Modalities Test OSU6162; (−)-OSU6162

PASAT; Paced Auditory Serial Addition Test RAVLT; Rey Auditory Verbal Learning Test

RHIFUQ; Rivermead Head Injury Follow Up Questionnaire RLS85; Reaction Level Scale

RPQ; Rivermead Post Concussion Symptoms Questionnaire sMRI; Structural Magnetic Resonance Imaging

TBI; Traumatic Brain Injury TE; Echo time

TR; Repetition time

WAIS-IV; Wechsler Adult Intelligence Scale, 4th_edition

(11)

Original papers

I. Berginström N, Nordström P, Schuit R, Nordström A. The Effects of (−)-OSU6162 on Chronic Fatigue in Patients With Traumatic Brain Injury: A Randomized Controlled Trial. Journal of Head Trauma Rehabilitation 2017; 32(2): E46-E54.

II. Berginström N, Nordström P, Ekman U, Eriksson J, Andersson M, Nyberg L, Nordström A. Using Functional Magnetic Resonance Imaging to Detect Chronic Fatigue in Patients With Previous Traumatic Brain Injury: Changes Linked to Altered Striato-Thalamic-Cortical Functioning. Journal of Head Trauma Rehabilitation 2018; 33(4): 266-274.

III. Berginström N, Nordström P, Ekman U, Eriksson J, Nyberg L, Nordström A. Pharmaco-fMRI in Patients with Traumatic Brain Injury: A Randomized Controlled Trial with the Monoaminergic Stabilizer (−)-OSU6162. Journal of Head Trauma Rehabilitation 2018. Published Ahead of Print. doi: 10.1097/HTR.0000000000000440.

IV. Berginström N, Nordström P, Nyberg L, Nordström A. White matter hyperintensities increases with traumatic brain injury severity: associations to neuropsychological performance and fatigue. Manuscript.

(12)

Enkel sammanfattning på svenska

Traumatisk skallskada (engelska traumatic brain injury; TBI) är en av de vanligaste anledningarna till funktions- och förmågebegränsningar hos människor, framför allt bland yngre personer. Många patienter, särskilt de med lätta skallskador, återhämtar sig fort, men vissa får besvär som blir långvariga eller bestående. Ett av de vanligaste symtomen är mental trötthet. Trots att det är så vanligt med trötthet efter skallskada, och trots att det påverkar dessa patienters dagliga liv i mycket stor omfattning, finns det idag inga standardiserade metoder för att mäta eller behandla detta symtom. I denna avhandling undersöks därför nya sätt att behandla och mäta/diagnosticera mental trötthet efter TBI.

I studie I undersöktes effekterna av ett nytt sorts läkemedel, den dopaminstabiliserande substansen OSU6162, på trötthet efter skallskada. Resultaten påvisade inga effekter av OSU6162 på tröttheten, som i denna studie undersöktes med självskattningsskalor och neuropsykologiska tester.

I studie II undersöktes huruvida funktionell magnetresonanstomografi (fMRI) kunde användas för att undersöka och diagnosticera trötthet hos patienter med TBI. Resultaten visade att tröttheten efter TBI verkar vara beroende på rubbningar i kommunikationen mellan hjärnregionerna striatum, thalamus och frontala cortex, samt att fMRI skulle kunna vara en möjlig teknik att använda för att diagnosticera denna trötthet.

Studie III undersökte huruvida OSU6162 påverkade hjärnaktivitet mätt med fMRI. Här påvisades att OSU6162 hade möjliga behandlingseffekter främst i regionerna occipitotemporala cortex och orbitofrontala cortex. Här verkade hjärnaktiviteten normaliseras för de som behandlades med OSU6162, men inte för de som behandlades med placebo, när jämförelser gjordes med friska försökspersoner. Att effekterna sågs just i dessa regioner kan förklara varför det inte syntes några effekter på trötthet i studie I, eftersom dessa ligger utanför de regioner som visade sig kritiska för trötthet i studie II.

I studie IV undersöktes om skador i hjärnans vita substans kan förklara den lägre kognitiva funktionen och tröttheten efter TBI. Resultaten visade att skador i den vita substansen var mycket vanligare hos patienter med TBI än hos friska försökspersoner, och att en högre skadegrad innebar större skador i den vita substansen. Däremot kunde inte fler skador i vitsubstansen förklara den lägre prestationen på olika neuropsykologiska test eller ökad trötthet hos patienter med TBI.

(13)

Sammanfattningsvis påvisar resultaten att OSU6162 inte har effekter på trötthet efter TBI vid denna dos och längd på behandling. För att mäta och diagnosticera trötthet efter TBI kan fMRI vara en lovande teknik. Det krävs dock mer forskning både vad gäller behandling och mätning/diagnosticering av detta för många så handikappande symtom.

(14)

Preface

For the last decade, which corresponds to the absolute majority of my clinical career as a psychologist, I have worked in the rehabilitation of patients with traumatic brain injuries at the Neurorehabilitation Clinic at the Northern University Hospital in Umeå. In this work, I have come to appreciate the complicated area of neurorehabilitation, and all aspects of the patient that need to be attended to. For me, this means that rehabilitation medicine is a field of many professionals, and the entire team consisting of physicians, physiotherapists, occupational therapists, social workers, nurses and others, is essential in this complex work.

As psychologists in neurorehabilitation, we take great pride in our ability to assess and detect subtle cognitive impairments in our patients using neuropsychological tests. Impairments that may be hard to understand by patients and their surroundings, but still enough to affect them in their everyday life, their social relationships and their ability to take part in leisure activities or work. This, together with the emotional consequences that often follow after a TBI, makes it a truly inspiring field to work in, a field where I as a psychologist really can make a difference in peoples’ lives.

Over the years, I have learned that some of these cognitive and emotional consequences are more or less common in this patient group. However, the absolute most common symptom that my patients with TBI complain about is fatigue. Even though I believe that I can capture this fatigue in some of my patients using neuropsychological tests, this is far from true for most of them. In a time where objective measures of symptoms are becoming more important, this is truly frustrating for me, and sometimes devastating for patients. Further, we have no way of treating this kind of fatigue in patients, even though the impact of fatigue on patients’ everyday life can be lessened by patients learning the importance of balance between activity and rest. These problems in my everyday clinic is what brought me into research on fatigue after TBI, because we certainly need to find new ways for treatment and diagnosis of this symptom that results in severe disability in so many people.

(15)

(16)

Introduction

Traumatic Brain Injury

Trauma is one of the most common causes of injury to the human brain, with more than 10 million people suffering a traumatic brain injury (TBI) globally each year.1_{Common reports of the incidence of TBI have been around}

200-300/100 000,2_{but the actual number of TBI may be as high as 600/100 000,}

since many mild TBIs are never treated at hospitals.3_{The fact that TBIs are so}

common, and that many patients do not seek medical attention and not become part of the official statistics, have prompted several authors to use the term “silent epidemic” when describing TBI.4-6_{Although the majority of TBIs is not fatal, TBI}

is the primary cause of death in adolescents and young adults.7

A TBI occurs when a mechanical force, direct or indirect, is applied to the brain. This causes an acceleration followed by a deceleration in the brain within the skull, which can cause both focal and diffuse damage. Focal damage includes subarachnoid and intraparenchymal haemorrhages, subdural and epidural hematoma, cerebral contusions, and later edema. These are all possible to detect with a computed tomography (CT) scan,8_{normally the first radiological exam}

performed on patients suspected to have suffered a TBI.9_{Diffuse damage include}

microvascular damage and diffuse axonal injury (DAI). In DAI, shearing forces causes a tearing between white and grey matter tissues, which causes axonal lesions in the subcortical white matter. These damages are not detectable on a CT scan, and are sometimes difficult to adequately describe after standard magnetic resonance imaging (MRI).10_{Focal and diffuse damages often coexist, especially}

in severe cases of TBI, affecting both morbidity and mortality.11_{Mild traumatic}

brain injuries might not always result in structural damage to the brain that is detectable on standard radiological examinations such as CT or MRI. Instead, the acute clinical symptoms, such as loss of consciousness, dizziness, or confusion, may be attributed to functional disturbances rather than structural damage in the brain.12

Severity of brain injury is determined on characteristics at the time point of injury. The most commonly used assessment scale is the Glasgow Coma scale (GCS13_{). This scale examines responses in the patient regarding eye-opening,}

motor and verbal responses. The patient gets a point in each of these categories for their best response, and receives a total score of 3-15. A total of 3-8 corresponds to a severe TBI, 9-12 to a moderate TBI, and 13-15 to a mild TBI.14_In

Sweden, it has been more common to use the Reaction Level Scale (RLS8515_),

which has shown high consistency with the GCS.16_{However, when an initial}

(17)

characteristics at the time of injury have been used to indicate severity. These include length of loss of consciousness, alteration of mental state, posttraumatic amnesia, or findings on structural neuroimaging.17_{Although there is a}

relationship between TBI severity and outcome, it is important to note that for the individual patient, the TBI severity does not always correspond to later impairments.18_{This is especially true for patients at the mild side of the}

continuum.19,20_{Thus, it has been proposed that in the later stages of TBI, current}

cognitive functioning, and not TBI severity, should be used as the basis for planning treatment and rehabilitation.21

Most patients with moderate or severe TBI experience long-term or persistent symtoms,22,23_{but the recovery prognosis in patients with mild TBI is more}

complicated. While a majority of patients suffering from mild TBI will recover within 3 to 6 months,24,25_{some will experience persisting and disabling symptoms}

for years to come.26,27_{This “miserable minority”}28_{seems to show higher}

injury-related stress, but also a worse premorbid emotional and physical health than those with better recovery.29_{Although there is a clear association between}

symptom severity in the first days or weeks after mild TBI, some may develop persistent symptoms in a later stage.30

The consequences reported by patients with TBI include a wide range of symptoms, of both physical, cognitive and emotional nature. These symptoms can be present in the acute, subacute, or chronic phase of the TBI. Physical symptoms include headaches, dizziness, and sensory disturbances including, but not limited to, light and noise sensitivity.31_{Common cognitive symptoms after}

TBI are deficits in attention and mental speed processing,32-34_long-term

memory,32_{working memory,}33_{and executive functions such as cognitive}

flexibility and inhibition.34_{Patients with TBI also display higher rate of emotional}

difficulties compared to the general population,35_{especially depression, anxiety,}

and increased symptoms of stress.36

Fatigue after traumatic brain injury - characterization and definition

Many patients in the chronic phase of TBI suffer from one or several of the symptoms described above, but one of the absolutely most common symptoms in this patient group is fatigue. Although studies vary in the prevalence of fatigue after TBI, around 70% of patients in the chronic phase of TBI report increased fatigue,32,37,38_{which by far exceeds the prevalence of fatigue in the general}

population, often reported to be around 10-20%.39,40_{Most improvement (i.e.}

reductions) in fatigue takes place during the first year after injury, with minimal improvements in the following years for those with persisting symptoms.41

(18)

but probably persists even longer as a chronic symptom. Fatigue is also common in several other disorders affecting the brain, such as stroke,43_{Parkinson’s}

disease,44_{and multiple sclerosis.}45_{Fatigue is therefore not an illness or a}

diagnostic entity in itself; instead it is a consequence of disturbances in the brain.46

The fatigue that patients experience after suffering a TBI is difficult to describe, since it is subjective in nature,47_{and has not been given a clear definition. It is}

possibly best described by the patients themselves, in Cantor et al47_{(p. 876):}

• “Anytime I have to focus . . . I’m fatigued.”

• “It’s like you disconnect – I can’t lift my arms, I just can’t – I’m not here so I have to go to sleep.”

• “Like sometimes I feel that I need to be alone, I have to be alone, yes, no more stimulation.”

• “Responding to noise and light and people, in a restaurant, I know, I have to sit on the end of the table and face the wall. Otherwise, I know that I have to leave the restaurant.”

• “Your brain gets full, so it is both a response but also what you can do… it’s inaccuracy in things that you used to be able to do.”

• “You lose what you were talking about, and you can’t hear what anybody else is saying either, and you are trying to grab that thought. It is totally exhausting and fatiguing and it changes your life. It just makes me want to cry thinking about it just because it drives you so easily, you just want to go lay down, you don’t want to participate anymore, you quit and a major part of that fatigue is that it changes how you react with everybody . . . but you lose it and its disconnecting on all levels, then you are frustrated with yourself.”

• “The thing about fatigue is that we all think of fatigue as going to sleep and being oh so tired. I would welcome, welcome feeling tired . . . mostly I wake up, head hurts, I’m not rested, those kinds of things”

This kind of fatigue is difficult to understand, both for the patients themselves and for people around them. Since many of the patients have no visible symptoms of the TBI, it is often hard to accept both for themselves and others that they have to live with “this extreme exhaustion which may appear suddenly, and without previous warning during mental activity, /…/ especially /…/ as the fatigue may appear even after seemingly trivial mental activities” (p. 49146_{). Around 30% of}

TBI patients with fatigue are regarded as lazy by their family.48_{Together with an}

often very long mental recovery time,49_{it is easy to understand that fatigue affect}

these patients’ lives deeply. Fatigue in the chronic phase of TBI has indeed been shown to be related to overall disability and the ability to take part in

(19)

activities,41,50_{impaired cognitive function,}51,52_{emotional disturbances,}37,51_and

decreased employment status.37,51,53_{Actually, fatigue has been shown to uniquely}

contribute to disability status after TBI, even when adjusting for injury severity, depression, and impairments in executive functions.54_{In addition, many patients}

with fatigue after TBI also suffer from sleep wake-disturbances. Fatigue might be an underlying stressor that is a precipitating factor behind insomnia after TBI,55

but other accompanying factors, such as anxiety or depression, might also be important underlying mechanisms.56_{Having trouble with sleep will of course}

increase the impact of fatigue in the patients’ everyday life.

It is not only in the everyday life of patients that fatigue is difficult to understand. Researchers have not been able to agree upon a clear scientific definition of fatigue after TBI. Some use what Chaudhuri and Behan57,58_{call central fatigue,}

defined as “the failure to initiate and/or sustain attentional tasks (‘mental fatigue’) and physical activities (‘physical fatigue’) requiring self-motivation (as opposed to external stimulation)”(p. 3557_{). This is different from peripheral}

fatigue, which is not related to mental abilities, but more strictly to neuromuscular output. Others have specified this further by pointing out the causes and defined fatigue as: “The awareness of a decreased capacity for physical and/or mental activity due to an imbalance in the availability, utilization, and/or restoration of resources needed to perform activity.” (p. 4659_{). However, there is}

not a direct relationship between fatigue after TBI and performance on either cognitive or physical tasks.47,60_{Thus, Kluger et al,}61_{in an attempt to propose a}

unified taxonomy for fatigue in patients with different kinds of neurological illnesses, distinguished between self-experienced fatigue and fatigability, where the latter is directly related to decreased performance on a given task.

In this thesis, the definition of fatigue is based on Chaudhuri and Behan’s term central fatigue, but with the Kluger et al distinction between fatigue and fatigability kept in mind. The definition of fatigue in this thesis is therefore: A failure to initiate and/or sustain attentional tasks or physical activities, or a self-experienced increase in fatigue that is not directly related to deterioration in performance in attentional tasks or physical activities.

Mechanisms behind fatigue after traumatic brain injury

Despite the prevalence of fatigue after TBI, and the impact it has on patients’ lives, the underlying mechanisms remain unclear. However, there are a few theories on the neurobiological basis of TBI-related fatigue. On the cellular level, it has been proposed that TBI causes a low-grade neuroinflammation affecting astrocytes, the supporting cells within the brain.62_{This inflammation affects the possibility}

(20)

glutamate levels are suggested to cause unspecific neuronal signaling and thus lack of energy.

On a systems level, Chaudhuri and Behan57,58_{specified the importance of}

dysfunction of the non-motor functions of the basal ganglia for fatigue in neurological disorders. According to their theory, disruptions within striato-thalamic-cortical loops may be of either structural, metabolic or neurochemical etiology, thus explaining why fatigue is common in so many different neurological disorders. Due to the importance of dopamine and serotonin within these loops, several studies have examined the potential impact of these neurotransmitters on fatigue. Evidence of relationships between increased levels of serotonin and fatigue have been found in patients with Parkinson’s disease,63_multiple

sclerosis,64_{and chronic fatigue syndrome.}65_{Others have stressed the relationship}

between the dopamine system and fatigue,66,67_{and even termed the “dopamine}

imbalance hypothesis” of fatigue in neurological disorders.68_{This hypothesis}

states that since either too high or too low levels of the neuromodulator dopamine in the central nervous system is associated with lower cognition, there is a need for balance in dopaminergic neurotransmission for optimal cognitive functioning. Although the “dopamine imbalance hypothesis” has been studied to some extent regarding fatigue in neurological disorders, more well-designed studies are needed to confirm this hypothesis.68

Many of the above-mentioned studies on the neuroanatomical basis of fatigue, both original studies and reviews, mentions that one single explanatory mechanism for such a multidimensional concept as fatigue is unlikely. Others theories include psychosocial,69_cognitive,70_{and genetic}71_{explanations of fatigue}

after TBI. Even though striato-thalamic-cortical loops seem to play an important part in fatigue after TBI, other factors might be of importance as well.

Assessment of fatigue after traumatic brain injury

Assessing fatigue after traumatic brain injury is not a straightforward task. Considering the differences between fatigue and fatigability as described above, there is a need to examine fatigue both regarding self-experience and the effects of fatigue on work output. Since fatigue after TBI mainly regards mental fatigue, this means studying cognitive performance.

There are over 30 self-assessment scales of fatigue,72_{but no “gold standard” exists}

for measuring fatigue in patients with TBI.47_{Many of the scales, such as the}

Fatigue Severity Scale (FSS73_{) and the Mental Fatigue Scale (MFS}49_{), have been}

found to have acceptable psychometric properties. These scales, and most other scales focusing on fatigue, captures what Genova et al74_{call “trait fatigue”. This}

(21)

such as “Fatigue causes frequent problems for me” (FSS) or “If you have to take a break, how long do you need to recover after you have worked ‘until you drop’ or are no longer able to concentrate on what you are doing?” (MFS). “Trait fatigue” should according to Genova et al74_{be distinguished from “state fatigue” which is}

a more transient condition, more able to fluctuate due to internal or external stimuli (such as rest or task occupancy). This kind of fatigue has to my knowledge only been assessed using simple ratings of fatigue on scales from 0-10 or with Visual Analog Scales (VAS). One major problem with using self-assessment is the subjective nature of these scales, in particular when examining patient groups where self-awareness deficits might be common, such as patients with TBI.75

Indeed, it has been recommended that interpretation of self-assessment of fatigue should be made with caution in patients with TBI, due to self-awareness deficits.75

Thus, to obtain a more objective measure of fatigue, several studies have used neuropsychological tests. Many studies have indeed demonstrated relationships between fatigue after TBI and neuropsychological measures, mainly attention and information processing speed.52,70,76-83_{When examining fatigue in patients}

with TBI, a certain focus on these functions is therefore important.56_Other

functions, such as working memory and executive functioning,82_{have also been}

shown to be related to fatigue after TBI. However, these results have not been thoroughly replicated, and the understanding of the relationship between cognitive performance and fatigue is still not fully understood.47,60_{This might be}

due to two reasons: 1. The difference between fatigue and fatigability as described by Kluger et al,61_{meaning that measuring fatigue subjectively (i.e. with}

self-assessment scales) and fatigability objectively (i.e. with neuropsychological tests) means that there are two different, although possibly related, constructs that are being measured; 2. The coping hypothesis,84_{which postulates that patients with}

TBI need to compensate for slower mental processing speed and attentional deficits by a constant extra (neuro-) physiological effort, which causes fatigue. Consequently, although patients with TBI that suffer from fatigue may perform as well as healthy controls on neuropsychological tests, the extra physiological effort needed to do this makes them fatigued. Thus, studying only level of performance, and/or only self-experienced fatigue, may not be sufficient when examining fatigue according to the coping hypothesis. Although some have used strictly physiological measures52_{when examining fatigue and cognitive functions}

in TBI, using functional neuroimaging might be one way of directly examining what is going on in the fatigued brain in individuals with a TBI.

(22)

Assessing fatigue after traumatic brain injury using functional magnetic resonance imaging

Functional magnetic resonance imaging (fMRI) uses the fact that neurons signaling need oxygen. Oxygenated and deoxygenated hemoglobin have different magnetic properties. In fMRI, the blood-oxygen-level dependent (BOLD) signal provides information about changes between oxygenated and deoxygenated hemoglobin during performance of a task as compared to a baseline condition. This information given by the BOLD signal can be interpreted as a measure of neural activity, although indirect.

Several studies have used fMRI to investigate cognitive and neurophysiological abnormalities after TBI, mainly within working memory paradigms. Two of the first studies in the field were conducted in the late 1990s by McAllister et al,85,86

with results showing mainly increased activations during working memory tasks within the frontal lobes in patients with TBI as compared to healthy controls. Thereafter, numerous studies have shown both regional hyper- and hypoactivation in patients with TBI when performing mental speed, attention, working memory, and long-term memory tasks (for a review, see Mayer87_{). In a}

meta-analysis, Bryer et al88_{showed that this discrepancy in results may be}

task-dependent, with hyperactivation in patients being more consistently observed in continuous or high load tasks, and hypoactivation in discrete or low load tasks. Bryer et al88_{interpreted hypoactivation as a result of patients with TBI being less}

engaged by the task, or that there is heterogeneity within the TBI sample in engagement of the task. Others have concluded that hypoactivation is due to failure in neural recruitment after injuries.89_{Hyperactivation has been explained}

as a normal response to increased load due to cognitive difficulties and neural inefficiency,90_{inhibitory difficulties,}91_{neural compensation,}85,86_{and effects of}

brain reorganization.92

Some fMRI studies with patients suffering from TBI have interpreted alterations in the BOLD signal as indicative of extra mental effort or fatigue.90,93-95_However,

at the beginning of this decade, only one fMRI study had explicitly examined fatigue in patients with TBI . Kohl and colleagues96_{used a sustained attention and}

processing speed task, the modified Symbol-Digit Modalities test (mSDMT), in an attempt to find neural correlates of mental fatigue. While there were no differences in task performance, patients with TBI showed increased activation in several regions over time during a 21-minute task. These regions included the superior parietal cortex, the middle frontal gyrus, the basal ganglia (caudate), and the anterior cingulate. Healthy controls showed decreased activity over time in the same regions. These results were in line with previous research from the same research group investigating fatigue in patients with multiple sclerosis.97_{With a}

theoretical basis in the coping hypothesis,84_{the authors interpreted increased}

(23)

ganglia was of specific interest in view of the theory that patients with neurological disorders and fatigue show dysfunction within striato-thalamic-cortical loops.57,63_{Unfortunately, neither Kohl et al}96_{nor Deluca et al}97_examined

fatigue in patients in any other way, such as with subjective self-report measures. During the recent years, a few other studies have investigated fatigue after TBI using fMRI. One study showed that fatigue after TBI was related to increased activation in caudate nucleus during a working memory task, but to decreased activation during the mSDMT processing speed task mentioned above.98_{At rest,}

the human brain at normally shows neural signaling within organized networks,99

often termed the default mode network.100_{fMRI studies of resting state}

connectivity have shown that disruptions in the connectivity between thalamus and middle frontal cortex at rest are related to fatigue in patients with TBI.101,102

Recent arterial spin labeling studies have found similar alterations to be related to fatigue after TBI.103,104_{In addition, several studies have examined fatigue in}

other neurological patient groups and shown that fatigue is related to alterations in striato-thalamic-cortical loops using both task105-108_{and rest fMRI.}109,110

According to the results from the studies summarized above, fMRI seems to offer a possibility to detect fatigue in patients with TBI or other neurological disorders. Still, functional MRI is rarely used in clinical practice in patients with TBI, while structural magnetic resonance imaging (sMRI) is very commonly used. sMRI uses different sequences to acquire high resolution images of the brain. These sequences have different properties to detect special features of abnormalities in the brain. The T1-weighted sequence return high resolution images with high grey-white matter contrast. The T2-weighted sequence can accurately establish presence of gliosis or edema within the brain, and characterize location or boundaries of cerebrospinal fluid. The gradient echo sequence is particularly sensitive in detecting hemosiderin depositions (residual byproducts after a bleeding). The fluid attenuated inversion recovery (FLAIR) is specifically sensitive in detecting pathologies within white matter. Abnormalities in white matter will show up as bright areas on the FLAIR image, and is thus called white matter hyperintensities (WMH). Studies on sMRI have revealed relationships between fatigue and structural brain lesions within striato-thalamo-cortical loops both in TBI111_{and other patient groups.}112-114_{Fatigability, but not self-assessed}

fatigue, was in a recent study115_{related to total lesion volume in white matter,}

indicating that a decrease in connectivity could be an important factor.

In summary, there seems to be some evidence that fatigue after TBI, and fatigue in other neurological disorders, is related to disturbances in striato-thalamic-cortical loops. However, the underlying mechanisms behind these alterations are not fully understood, neither exactly how these alterations relate to fatigue. Thus,

(24)

these alterations and to detect or diagnose fatigue in these patients are needed. Further, these previous studies give no clear indication how fatigue should be treated.

Treatment of fatigue after traumatic brain injury

Due to the difficulties in the reliable assessment of fatigue after TBI, together with an incomplete understanding of the underlying mechanisms, finding treatments for this disabling symptom have been difficult. All systematic reviews on treatments of fatigue after TBI during the last five years conclude that there are no treatments to be recommended, neither pharmacological nor behavioral, due to a lack of conclusive scientific evidence.116-120_{Treating other symptoms and}

disorders (e.g. depression, anxiety, pain, sleep-wake disturbances) that might increase the fatigue, and finding a balance between rest and activity, is today the best recommendation for patients with fatigue after TBI.56,121_{However, there are}

several promising treatments that should be studied further.

Behavioral interventions of fatigue after traumatic brain injury There are few studies that have examined the effects of different behavioral interventions after TBI and used fatigue as primary outcome. However, several studies have examined fatigue as secondary outcome of the intervention. At least six studies have examined the effects of cognitive behavioral therapy (CBT) in patients with TBI and used fatigue as secondary outcomes. Three case studies examined the effects of CBT on insomnia.122-124_{All of these showed indications of}

effects on fatigue, but the effects were generally weak, and difficult to interpret due to the study designs. Other CBT studies for social anxiety,125_{post-concussion}

symptoms,126_{and depression after TBI,}127_{also revealed effects on fatigue,}

although this was not the primary target. Johansson and colleagues performed two studies128,129_{examining the effects of Mindfulness-based stress reduction}

aimed at reducing fatigue after stroke or TBI. They found effects on fatigue of both person-to-person, and internet delivered mindfulness, possibly with the latter being superior. These promising results of CBT inspired Nguyen and colleagues130_{to design an adapted form of CBT treatment, specifically aimed at}

sleep disturbances and fatigue after TBI. Their initial pilot study of this therapy programme showed effects both on sleep quality, depression and fatigue. In a similar fashion, Raina et al131_{developed an internet-based manualized CBT}

intervention combining psychoeducation and problem-solving therapy targeting fatigue management after TBI. With small to medium effect sizes on several fatigue measures, the authors found effects of the intervention in a small TBI sample. It should be made clear that there is a need for replication of the results from all of these studies, especially including larger samples and using more robust study designs, before any definitive conclusions can be drawn.

(25)

There have also been some studies on different physical exercise regimes after TBI that have included fatigue as secondary outcome. While some studies have not found any effects on mental fatigue,132-136_{others have found effects on both}

physical fatigability and mental fatigue.137,138_{Several other interventions such as}

working memory training,139_{electroencephalographic biofeedback,}140_light

therapy,141_{and cranial electrotherapy stimulation}142_{have indicated effects on}

fatigue, but these results have not been replicated.

Pharmacological interventions of fatigue after traumatic brain injury

There have been a number of studies that have tested different pharmacological interventions with fatigue as primary target in patients with TBI. These can broadly be divided on basis of the kind of drug: Modafinil, Methylphenidate, other interventions, and OSU6162.

Of three randomized, double-blinded, placebo-controlled trials of modafinil, 143-145_{all found effects on sleep quality or daytime sleepiness, but only one found}

effects on fatigue in patients with TBI.145_{Thus, treating sleep effectively does not}

seem to be enough to alleviate fatigue in these patients.

One randomized, double-blinded placebo-controlled study of methylphenidate found effects both on fatigue and fatigability in patients with TBI.146_Two

studies147,148_{of methylphenidate that used randomized, unblinded cross-over}

designs found significant decreases in mental fatigue and increases in information processing speed after four weeks of treatment. These positive effects were dose-dependent, as patients with higher (or normal) doses of methylphenidate (60 mg/day) showed more improvement than those with low doses (15 mg/day). The improvements were better than for patients receiving no treatment. The same research group also showed long-term effects of treatment with methylphenidate on both mental fatigue and several cognitive functions using a pre-post assessment observational design.149

Additional studies have evaluated other pharmacological interventions such as donepezil,150_piracetam,151_{and recombinant human growth hormone,}152_and

included fatigue as secondary outcome. These studies have had mixed results, and often suffered from low sample size and flawed study designs, such as unblinded pre-post designs.

In summary, there have been a number of both behavioral and pharmacological interventions evaluated for treatment of fatigue after traumatic brain injury, but due to mixed results, possibly reflecting poor study design quality and small samples sizes, there are still no treatments with reasonable scientific support

(26)

available for this disabling symptom. Thus, there is a need for more trials examining the effects of different forms of treatment, both previously tried and novel forms.

(−)-OSU6162

A novel chemical compound is the OSU6162. OSU6162 has been classified as a monoaminergic stabilizer, which stems from the fact that the direction of response to treatment with OSU6162 is dependent on dopaminergic tone.153_It

has been shown to affect both motor systems154_{and cognition}153_{in rodents,}

displaying these stabilizing properties. OSU6162 is well tolerated in healthy human subjects, and causes only minor side effects at high doses, including small elevations of heart rate and serum prolactin levels.155_{The compound has shown}

affinity to mainly D2/D3 receptors,156,157_{binding to the allosteric site for}

stimulation and dopamine release, and to the orthosteric site for inhibition.158_It

has also shown a possible affinity to serotonergic 5-HT2A receptors,157,159_thus

potentially affecting both dopaminergic and serotonergic systems. Since both these systems, and the homeostasis within them, have been shown to influence fatigue after TBI,68,160_{there have been a few studies examining whether OSU6162}

can alleviate fatigue in neurological patient groups.

One study used a non-randomized, crossover design to test the tolerability and efficacy of OSU6162 in 15 patients with Huntington’s disease.161_{The results}

revealed effects on one item (‘vitality’) of the Short-Form 36 Questionnaire, but no other treatment effects were seen, including different cognitive tests and self-assessment scales. In a recent randomized double-blind placebo-controlled study of OSU6162 in 62 patients with Chronic Fatigue Syndrome, there were no effects when examining the entire group, but in a subsample of patients, with concomitant pharmacological treatment for depression, OSU6162 did have effects on fatigue.162_{Furthermore, in an open-label single-arm study, OSU6162}

showed effects on mental fatigue and depression in 30 patients with multiple sclerosis.163

Only one study has examined the effects of OSU6162 on fatigue in patients with TBI. In a randomized crossover, double-blind and placebo-controlled pilot study, Johansson et al164_{included 6 patients with TBI and 6 patients with stroke, all}

suffering from fatigue. These patients were treated with OSU6162 or placebo in increasing doses from 15 to 45 mg twice daily over four weeks. Patients receiving OSU6162 showed improvements on both self-assessment of mental fatigue, and trends towards improvements on neuropsychological tests of attention and processing speed. This latter study was the inspiration for the clinical trial of OSU6162 (Studies I and III) that forms the basis of this thesis.

(27)

Thesis Rationale and Aims

Due to the prevalence of fatigue in patients with persisting symptoms after TBI, and the limited understanding of mechanisms behind fatigue after TBI and how it should be assessed and treated, the aims of this thesis were:

1. To evaluate the effects of monoaminergic stabilizer OSU6162 on mental fatigue, as assessed by self-assessment scales and neuropsychological tests, in patients in the chronic phase of TBI (Study I).

2. To evaluate whether fMRI BOLD signal could be used to detect and diagnose fatigue in patients in the chronic phase of TBI (study II). 3. To examine the effects of OSU6162 on fMRI BOLD signal in patients in

the chronic phase of TBI (Study III).

4. To examine whether white matter hyperintensities (WMH) in patients in the chronic phase of TBI may influence cognitive function and fatigue (Study IV).

(28)

Materials and Methods

Study Designs

The studies in the thesis were approved by the Regional Ethical Review Board in Umeå (dnr 2011-346-31M, with supplement dnr 2015-87-32M), and the Swedish Medical Products Agency. The studies were performed in accordance with the principles stated in the World Medical Association’s Declaration of Helsinki. The clinical randomized trials (Study I and III) were conducted according to the study protocol (EudraCT clinical trial registration no. 2011-004990-10).

Studies I and III were randomized double-blinded placebo-controlled clinical trials of the effects of OSU6162. In study I, the outcome was measured with self-assessment scales and neuropsychological tests. In study III, outcome was measured using fMRI.

Studies II and IV were cross-sectional studies analyzing differences in fMRI-data (Study II) and structural MRI-data (Study IV) in patients with different severities of TBI and healthy controls.

Participants and Inclusion Procedure

All patients with TBI were the same for Studies I-IV, and were thus included through the same inclusion process. The patients were recruited using registers over all 490 patients with ICD-10165_{diagnostic codes S06.x (intracranial injury),}

F07.2 (postconcussional syndrome), or T90.5 (sequelae of intracranial injury) that had visited the Neurorehabilitation Clinic in Umeå during 2006 to 2013. The inclusion criteria were:

1. Suffered a Traumatic brain injury > 12 months ago. 2. Age 18 to 65.

3. Moderate disability or better recovery (i.e. > 5) on the Extended Glasgow Outcome Scale.166_{This means that patients must be able to take care of}

themselves in their home and be able to travel and go grocery shopping alone. Still they can have impairments in their ability to work or take part in leisure activities.

4. Self-experienced fatigue, defined as a score > 36 on the Fatigue Severity Scale.73

Exclusion criteria were:

1. Other psychiatric or neurological diseases such as severe depression and anxiety, bipolar disease, obsessive-compulsive disorder, psychosis and addiction to alcohol or drugs.

(29)

2. Sick sinus syndrome; resting heart rate below 50 beats per minute; congestive heart failure classified as functional Class III or IV by the New York Heart Association;167_{myocardial infarction within six months of}

randomization; a prolonged corrected QT at screen or pretreatment (defined as a corrected QT interval of > 450 milliseconds for males or > 470 milliseconds for females); other clinically significant heart conditions which would negatively impact on the patient completing the study.

3. Clinically significant liver disease and/or an elevation in either total bilirubin, alkaline phosphatase, lactate dehydrogenase or serum glutamic-oxaloacetic transaminase of > 2 times the laboratory reference. 4. Clinically significant renal disease and/or an elevation in serum

creatinine of > 1.5 times the laboratory reference. 5. Presence of active neoplastic disease.

6. Electroconvulsive therapy within the last 90 days.

7. Surgical or medical conditions, which, in the judgment of the principal clinical investigator, might have interfered with the absorption, distribution, metabolism or excretion of the drug.

8. Drug treatments capable of inducing hepatic enzyme metabolism (e.g., barbiturates, rifampicin, carbamazepine, phenytoin, primidone) within the previous 30 days of enrollment in this study (or 5 half-lives of inducing agent, whichever longer).

9. Personal or immediate family medical history of seizures. 10. Clozapine treatment.

11. Unstable therapies were not allowed, but stable therapies were. A stable therapy was defined as having started at least 6 months before the study and continued to be unchanged during the study period. Examples of such medications are anti-depressant therapy such as citalopram (highest allowed dose 40 mg/daily), mirtazapine (highest allowed dose 45 mg daily) or sertraline (highest allowed dose 100 mg/daily). Other stable therapies with hypnotics and anxiolytics were also allowed if given at doses recommended by the manufacturers. Analgesics such as nonsteroidal anti-inflammatory drugs, acetyl salicylic acid and paracetamol were permitted as well as stable anti-hypertensive therapy. 12. Pregnancy

13. Women of childbearing age not on contraceptives.

14. Abnormal laboratory parameters: such as hemoglobin, white blood cells count, electrolytes, thyroid-stimulating hormone, thyroxine, vitamin B12 or folic acid above the laboratory references level.

15. Severe dementia.

(30)

After reading of medical records, 278 patients were excluded due to fulfilling one or more of the exclusion criteria, or residing outside the county of Västerbotten. 212 patients received an initial phone call, at the time of which 129 patients were excluded, mainly due to not experiencing fatigue. After another 13 patients who initially agreed to participate declined participation before baseline, a total of 70 patients performed all baseline assessments, excluding MRI/fMRI examination. Three of these patients were excluded on basis of the exclusion criteria, and one was excluded due to not suffering from fatigue. Another two patients dropped out after the baseline examination. Thus, a total of 64 patients were randomized to receive either OSU6162 (n = 33) or placebo (n = 31). See figure 1 for a summary of the inclusion process (also, see Figure 1 in Study I for a more detailed flow chart on reasons for exclusion). TBI severity was based on physician’s diagnosis based on self-reported or medical journal confirmed loss of consciousness in accordance with VA/DoD Clinical Practice Guideline for the Management of Concussion-Mild Traumatic Brain Injury.17

Figure 1. Flow chart for inclusion of patients with TBI to all four studies.

To investigate differences in functional and structural brain imaging between patients with TBI and people with no known brain injury, healthy controls were recruited for studies II-IV. These healthy controls were friends and families of patients that volunteered to participate. The inclusion and exclusion criteria were the same as for patients, but healthy controls were also excluded if they had suffered a concussion or more severe TBI. A total of 57 healthy controls reported interest, but to have groups with approximately equal size, only 30 healthy controls were included. These were chosen from the initial 57 to match the patient groups as closely as possible on age, gender, and education. Three of these healthy controls were excluded after performing all examinations, two due to scoring above cut-offs on self-assessment scales for clinical conditions, and one due to incidental findings during MRI/fMRI examination. Thus, a total of 27 healthy controls were included in the analyses in studies II-IV.

Procedure

At the beginning of the study, all patients received written and oral information regarding the study from the principal investigator and head physician, and all signed informed consent. Patients then went through medical examinations, electrocardiography (ECG), vital signs assessment and blood samples collection. After this, they completed self-assessment scales and went through

490 Eligible in

medical records 212 received phone call 70 examined at baseline

64 Randomly assigned at treatment start 4 excluded 2 drop-outs 129 Excluded 13 declined before enrollment 278 excluded

(31)

neuropsychological testing. If no reason for exclusion appeared during this first visit, the patient returned one week later for the MRI/fMRI examination, randomization to OSU6162 or placebo, and to start treatment. The treatment duration was 28 days, and the treatment group received OSU6162 in increasing dosage, starting with 5 mg twice daily during week one, 10 mg twice daily during week two, and 15 mg twice daily during weeks 3 and 4. To ensure the double blinding, the placebo group received the placebo in a similar pseudo-increasing dosage. All patients visited the clinic once weekly during the entire treatment period to meet with the principal investigator, go through medical examinations, vital signs assessment and to complete self-assessment scales. These weekly examinations were performed primarily for safety, and was not included in the outcome analyses in any study. On the last day of treatment (day 28), all patients returned and performed all examinations again, starting with the MRI/fMRI session, followed by medical assessments, self-assessment scales and neuropsychological testing. For illustration of the examination and treatment procedure, see figure 2.

Figure 2. Flow chart for the examination and treatment procedure for patients included in the clinical trial. Superscript Roman numerals indicate in which studies the examinations have been used as outcomes.

Healthy controls went through all examinations at one time point only, and performed all tests on the same day. After receiving written and oral information regarding the study and signing informed consent, they first went through MRI/fMRI examination, had a 30-minute break, went through neuropsychological testing and ended the day completing self-assessment scales.

(32)

Instruments

Self-Assessment Scales

To capture “trait fatigue”, two self-assessment scales of fatigue were used: The Fatigue Severity Scale (FSS73_{) and the Mental Fatigue Scale (MFS}49_{). Both scales}

focus on fatigue, but while the former is more focused on physical symptoms of fatigue and consequences in everyday life, the latter focuses on mental or cognitive fatigue and includes consequences on emotional functioning. The FSS is a 9 item Likert scale, with responses on each question ranging from 1 to 7. Mean scores of 4 or above (or a total of > 36) have been used to indicate severe fatigue,73

but later studies indicate that the cutoff should be set higher to avoid overdiagnosing.40_{The scale shows high internal consistency and validity for}

fatigue in patients with TBI.81,168,169_{Although less well studied than the FSS, the}

MFS also shows high internal concistency,49_{and has been validated towards}

fatigue in patients with TBI.170_{This scale consists of 15 questions rated on a scale}

from 0 to 3. Questions incorporate both emotional, cognitive and sensory aspects of fatigue. A cut-off of 10.5 have been suggested to indicate problems with mental fatigue.170

The participants also completed the Hospital Anxiety and Depression Scale (HAD171_{), a common measure of depression and anxiety in patients with somatic}

disorders, including TBI.172_{Lastly, all participants completed the Rivermead Post}

Concussion Symptoms Questionnaire (RPQ173_{) and the Rivermead Head Injury}

Follow Up Questionnaire (RHIFUQ174_{). These scales capture common symptoms}

after concussions and more severe head injuries (RPQ), and changes from before to after the injury in daily activities, such as the ability to participate in conversations or to perform leisure or work-related activities (RHIFUQ).

Neuropsychological tests

All participants underwent an extensive neuropsychological test battery. The tests were given in the following order:

• Rey Auditory Verbal Learning Test (RAVLT175_{) This is a test of verbal}

learning and memory. It consists of five trials of learning, one distraction list, short term recall and long-term recall. The long-term recall part of this test was performed after the Trail Making Test below.

• Coding from Wechsler Adult Intelligence Scale, 4th_{edition (WAIS-IV}176₎

measures processing speed.

• Digit Span from Wechsler Memory Scale, 3rd_{edition (WMS-III}177_{) is a test}

of auditory attention and working memory.

• Trail Making Test from Delis-Kaplan Executive Functions System (D-KEFS178_{) consists of four trials of visual scanning, number sequence,}

(33)

letter sequence, and number-letter switching, and measures visual search ability, attention, and the executive function switching.

• Paced Auditory Serial Addition Test (PASAT179_{) measures information}

processing ability, both quantity and speed.

• Symbol Search from WAIS-IV176_{measures processing speed.}

• Block Span from WMS-III177_{is a test of visual attention and working}

memory.

• Color-Word Interference Test from D-KEFS178_{primarily measures the}

executive functions of inhibition and switching.

• Verbal Fluency from D-KEFS178_{is also a test of executive functions,}

primarily word fluency and switching.

• The computerized Integrated Visual and Auditory Continuous Performance Test (IVA180_{), which measures sustained attention and}

response inhibition. This last test was performed after participants had completed the self-assessment scales, and not as a part of the rest of the neuropsychological test regime.

Structural and functional Magnetic Resonance Imaging

For studies II and III, fMRI was the main outcome measure, and for study IV, structural MRI imaging was the variable of interest. The same MRI equipment and procedure were used for all patients with TBI and healthy participants on all scanning occasions.

MRI Imaging procedure

The scannings were performed on the same 3T Discovery MR 750 General Electric scanner (General Electric Company, Chicago, Illinois, USA, Figure 3).

(34)

Figure 3. The 3T Discovery MR 750 General Electric scanner used for all scannings in studies II-IV. Photo: Mikael Stiernstedt

The fMRI sequence was performed first. In this sequence, BOLD-contrast sensitive T2-weighted single-shot gradient echo-planar imaging sequences were used. These were collected as 37 transaxial slices, each with a slice thickness of 3.4 mm, with 0.5 mm spacing and an Echo time (TE) of 30 ms, Repetition time (TR) of 2000 ms, flip angle of 80°, and field of view of 25 × 25 cm. The fMRI sequence was followed by the anatomical image acquisition. T1-weighted images were collected using a 3D fast spoiled gradient-echo sequence with 176 slices, and a slice thickness of 1 mm; TR = 8.2 ms, TE = 3.2 ms, flip angle = 12° and field of view = 25×25 cm. Fluid attenuated inversion recovery (FLAIR; used in study IV) images were acquired using a 2D T2 FLAIR sequence in 48 slices with 3 mm thickness; TR: 8000 ms, TE: 120 ms, field of view: 24x24 cm.

In the preprocessing stage, all fMRI data were slice-time and motion corrected, followed by coregistration to the T1 image. The T1 images were segmented, and a Dartel181_{template was created for each participant, which was used for}

normalization in Montreal Neurological Institute (MNI) space. Smoothing were performed using a Gaussian filter with full width half maximum of 8 mm. All structural images were reviewed by a clinical radiologist to examine the extent of damage in patients with TBI, and to discover possible abnormalities in healthy controls.

(35)

fMRI task – The modified Symbol Digit Modalities Test

During fMRI scanning, participants performed a 27-minute fatiguing attention and processing speed task called the modified Symbol Digit Modalities Test (mSDMT), which have been used in several previous studies to assess fatigue in patients with neurological disorders.96,97,182_{The task was programmed and}

administered using E-prime software (version 2.0.10.353, Professional Psychology Software Tools, Inc, Pittsburgh: www.pstnet.com/eprime). In this task, participants viewed a digit-symbol code-key, with digits 1-9 paired with a specific symbol (Figure 4). Below this code-key, one symbol-digit pair appeared. Participants were supposed to click a response button on an MRI-compatible keypad (Current Designs package 932, Current Designs, Philadelphia, Pennsylvania) with their right index finger if the pair matched a pair in the code key, and with their left index finger if it did not. The code-key changed after each trial to avoid learning effects. The trial was shown until a response was given, or until 6 seconds had elapsed. After this, a randomly varying inter-trial-interval passed until the next trial began. These intervals were of 0, 4, 8 or 12 seconds in length, with a fixation cross on the screen during this time. The test consisted of a total of 192 trials, lasting approximately 27 minutes. The participants practiced this task on a laptop before entering the scanner, to make sure that they had understood the task. This practice continued until participants answered at least five consecutive trials correctly.

Figure 4. Description of the modified Symbo0l Digit Modalities Test as administered to participants in studies II and III. Trial on the left displays a matching trial, and trial on the right displays a non-matching trial. ITI = Inter-trial-interval; s = seconds.

Assessment of “state fatigue” before and after fMRI task

To capture how this task affected the “state fatigue” of participants, they had to rate their fatigue directly before and after performing the task, while lying in the scanner. Participants were asked to rate their fatigue at that precise moment on a scale from 0 to 10, where 0 indicated no fatigue at all, and 10 indicated the worst possible fatigue.

(36)

Other examinations

For safety, and for sensitivity analyses in study I, patients with TBI in the clinical trial was examined medically with ECG, vital signs assessment and blood samples were drawn, at baseline and at treatment end. The blood samples were analyzed in regards to hormonal function (prolactin), pancreatic function (serum amylase, glucose), kidney/renal function (uric acid, urea nitrogen, creatinine), liver function (total protein, albumin, bilirubin, aspartate transaminase, alanine transaminase, alkaline- phosphatase, gamma-glutamyl transferase), serum electrolyte changes (Sodium and Potassium) and cholesterol and folic acid. Further, in study I, plasma concentrations of OSU6162 were evaluated in the treatment group. This was done by using liquid chromatography–tandem mass spectrometry, with a method previously described.156

Statistics

All analyses of statistical data that were not fMRI data, were analyzed using SPSS statistics IBM Corporation, Armonk, New York) versions 22 (studies I and II) or 24 (studies III and IV). The alpha-level was generally set to 0.05 in all analyses, except for fMRI-data. If not otherwise specified, differences between groups were tested using independent t-tests for continuous variables and Chi2_{-tests for}

categorical variables. fMRI data were analyzed using SPM 12183_{or an inhouse}

batch program called DataZ.

Study I

To examine effects of treatment, differences between pre- and post-treatment assessments were tested in linear regressions with treatment group as explanatory variable. These linear regression models were later performed adjusting for several covariates (gender, age, education, employment status, time since injury, and pathologic MRI/CT findings). Within-group differences from before to after treatment were tested using paired-samples t-tests. This study used an intention-to-treat protocol, where missing observations at the post-treatment assessment were compensated by the last observation carried forward method.

Study II

fMRI

To investigate whether there were any time effects on both fMRI BOLD signal and performance of the fMRI task, the fMRI session was divided into three parts. Part 1 corresponded to the first 64 trials of the task, Part 2 to trials 65-128, and Part 3 to trials 129-192.

(37)

To analyze fMRI data, a regression analysis was set up in SPM12, with a relative masking threshold of 0.15 and a high pass filter of 130 seconds. To adjust for serial correlations, an autoregressive model was used. 6 regressors were defined, 3 for experimental trials (Part1, Part2 and Part3) of the task, and 3 for the corresponding inter-trial-intervals (Cross1, Cross2, Cross3), and then convolved with the hemodynamic response. For each individual, two contrasts were set up: One for the entire task (task effect; Part 1+Part2+Part3-Cross1-Cross2-Cross3), and one for time effects (Part1-Cross1)-(Part3-Cross3). These two contrasts were then analyzed and compared between healthy controls and patients with TBI using two-sample t-tests. This comparison was done with a whole-brain approach using the family-wise error method (FWE) to control for multiple comparisons, with p < 0.05 (cluster size > 10). This analysis was later adjusted for differences in reaction time on the fMRI task and in presence of depressive symptoms. Subsequent analyses for the time effects used an uncorrected p < 0.005, in on beforehand hypothesized regions of the basal ganglia (caudate nucleus and putamen) thalamus, cingulate cortex and middle frontal gyrus. The same statistical threshold was used for correlational analyses between fMRI data and self-assessment of fatigue.

Behavioral data

Analyses comparing healthy controls and patients with TBI on self-assessment scales, neuropsychological tests and results the fMRI task (mSDMT) used ANCOVAs with education as covariate due to differences in this variable and its known effects on neuropsychological tests. Repeated measures ANOVA were used when examining differences between groups in “state fatigue” from before to after performance of the mSDMT (2x2, group vs time), and for testing differences between groups in reaction time and accuracy on the mSDMT changes during parts 1, 2 and 3 of the task (2x3, group vs time).

Study III

In Study III, the same fMRI contrast as in Study II (task effect) was used, one for the pre-treatment and one for the post-treatment examination. To evaluate the effects of treatment on changes in these contrasts from before to after treatment, a 2X2 (group by time) repeated measures ANOVA was used. An initial whole brain FWE-controlled analysis was followed by a whole brain uncorrected analyses at p < 0.001 and cluster size > 10. To test differences of contrast values in clusters with treatment effects between patients participating in the trial and healthy controls, independent samples t-tests were used. To examine treatment effects on behavioral data, a 2X2 (group by time) repeated measures ANOVA was used. It is important to note that in this study, in contrast to study I, the last observation carried forward method was not used.